Peptides play pivotal roles as the molecules for regulating various functions in vivo such as metabolism, growth, reproduction, maintenance of homeostasis, mental activities, biological protection or the like. These peptides are coupled to specific receptors on the cell membrane to transduce their information. So far, most of these physiologically active peptides have been isolated from tissue extracts, etc. based on their physiological activities followed by determination of their structures. Using these receptors, physiologically active peptides have recently been isolated from tissue extracts, etc.
On the other hand, the latest rapid progress of sequencing of genome or cDNA has made accessible to enormous information on DNAs. It is assumed that these DNAs would comprise the DNA encoding for physiologically active peptides hitherto unknown. However, most physiologically active peptides have only very short amino acid sequences. Therefore, even if one attempts to explore, from genomic DNA sequences or expressed sequence tag (EST), such unknown physiologically active peptides bearing a sequence in part similar to or a common motif to known physiologically active peptides, desired sequences similar to these known peptides are frequently found only in protein genes not at all associated with physiologically active peptides or in DNA sequences of non-translational region. It was thus extremely difficult to ascertain which the objective physiologically active peptide is in these peptides.
FMRF amide, one of physiologically active peptides, is a peptide isolated from the ganglia of bivalve, which structure was determined for the first time (Price, D. A. & Greenberg, M. J., Science, 197 670–671, 1977). Since then it has turned out that peptides having an RF amide structure at the C terminus and peptides having a structure similar to the RF amide structure are present over many species of the invertebrate animal. Many peptides having the RF amide structure are reported to be present especially in the nematodes. It is also known that most of these peptides are borne on one gene in such a state that a plurality of peptides is contiguous (Nelson, L. S., et al., Molecular Brain Research, 58, 103–111, 1998).
Turning to the vertebrate animal, LPLRF amide was isolated from the brain of chicken and identified to be an FMRF amide-like peptide having the RF amide structure. However, its gene structure remains yet unknown (Dockray, G. J., et al., Nature, 305, 328–330, 1983). In fish, C-RFa was recently reported to be a peptide with the RF amide structure. As peptides containing the RF amide structure in mammal, there are known two peptides purified and isolated from bovine (Yang, H.-Y. T., et al., Proc. Natl. Acad. Sci. USA, 82, 7757–7761, 1985) and neuropeptide SF (NSF) and neuropeptide AF(NAF) isolated from human cDNA, which are considered to correspond to the two peptides above. Recently, the present inventors identified prolactin-releasing peptides (PrRP) containing the RF amide structure in human, bovine and rats (Hinuma, S., et al., Nature, 393, 272–276, 1998).
Various reports have been published on the physiological activities of the FMRF amide peptides, which include, for example, acceleration or suppression of heartbeats, contraction or relaxation of various radular muscle, visceral muscle and retractor muscle, and hyperpolarization or depolarization of nerve cells. With respect to PrRP and LPLRF amides, prolactin-releasing stimulation activity, and nerve cell-stimulating effects or hypertension effects are reported, respectively.
As stated above, many important physiological activities have been reported on the RF amide structure-bearing peptides. However, it is totally unknown if there is any other peptide containing the RF amide or the like structure in mammals, except NSF, NAF or PrRP.
On the other hand, a variety of physiologically active substances such as hormones, neurotransmitters, etc. regulate the functions in vivo through specific receptor proteins located in a cell membrane. Many of these receptor proteins are coupled with guanine nucleotide-binding protein (hereinafter sometimes referred to as G protein) and mediate the intracellular signal transduction via activation of G protein. These receptor proteins possess the common structure, i.e. seven transmembrane domains and are thus collectively referred to as G protein-coupled receptors or seven-transmembrane receptors (7TMR).
G protein-coupled receptor proteins present on the cell surface of each functional cells and organs in the body, and play important physiological roles as the targets of molecules that regulate the functions of the cells and organs, e.g., hormones, neurotransmitters, physiologically active substances and the like. Receptors transmit signals into cells via binding with physiologically active substances, and the signals induce various reactions such as activation and inhibition of the cells.
To clarify the relationship between substances that regulate complex biological functions in various cells and organs and their specific receptor proteins, in particular, G protein-coupled receptor proteins would elucidate the functional mechanisms in various cells and organs in the body to provide a very important means for development of drugs closely associated with the functions.
For example, in various organs, their physiological functions are controlled in vivo through regulation by many hormones, hormone-like substances, neurotransmitters or physiologically active substances. In particular, physiologically active substances are found in numerous sites of the body and regulate the physiological functions through their corresponding receptor proteins. However, it is supposed that many unknown hormones, neurotransmitters or other physiologically active substances still exist in the body and, as for their receptor proteins, many of such proteins have not yet been reported. In addition, it is still unknown if there are subtypes of known receptor proteins.
One of the pathways to modulate biological functions mediated by the interactions of hormones or neurotransmitters with G protein-coupled receptors is the hypothalamus-pituitary system. In this system, the secretion of pituitary hormones from the pituitary gland is regulated by hypothalamic hormones (pituitatropic hormones), and the functions of the target cells/organs are regulated by the pituitary hormones released into the blood. Functions, which are important for the living body, including the maintenance of homeostasis and the control of development, metabolism and growth of a genital system and an individual organism, are regulated through this pathway.
The secretion of pituitary hormones is controlled by a positive feedback or a negative feedback mechanism by the hypothalamic hormone and the peripheral hormone secreted from the target endocrine glands.
It is also widely known that these hormones and factors as well as their receptors are not localized in the hypothalamus-pituitary system but are broadly distributed in the brain. This fact suggests that the substances called hypothalamic hormones are functioning as neurotransmitters or neuromodulators in the central nervous system.
Moreover, these substances are distributed even in the peripheral tissues as well and thought to be playing the role of important functions in the respective tissues.
It is also very important for development of drugs to clarify the relationship between substances that regulate elaborate functions in vivo and their specific receptor proteins. Furthermore, for efficient screening of agonists and antagonists to receptor proteins in development of drugs, it is required to clarify functional mechanisms of receptor protein genes expressed in vivo and express the genes in an appropriate expression system.
In recent years, random analysis of cDNA sequences has been actively studied as a means for analyzing genes expressed in vivo. The sequences of cDNA fragments thus obtained have been registered on and published to databases as Expressed Sequence Tag (EST). However, since many ESTs contain sequence information only, it is difficult to deduce their functions from the information.
It has thus been desired to find an unknown polypeptide (peptide) having RF amide-like structure or an unknown G protein-coupled receptor protein and using these peptides to develop a drug for the prevention, treatment or diagnosis for disease, comprising a novel physiologically active peptide, in particular, for regulating the secretion of pituitary hormones from the pituitary gland by regulating the secretion of hypothalamic hormones.